Apoptosis regulates notochord development in Xenopus

AbstractThe notochord is the defining characteristic of the chordate embryo and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos, however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that, by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirrors that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior–posterior axis, and its role in this process is discussed

UnIX-CARE: Universal Interface & Experience Via Collaborative Archive Repository Express

It is feasible to simplify interface design for better user experience without web developing skillfully. CARE or collaborative archive repository express holds the answer to universal interface & experience (UnIX) through algorithmic machine learning. CARE in collaboration with DATA and wiseCIO as a whole establishes a CMD triad for content management and delivery that harnesses rapid prototyping for user interface and propels user-centric experience by cohesive assembly of Anything as a Service (XaaS). Basically, user-centric experience makes a user centered without often webpage swapping while browsing in hierarchical depth via “In-&-Out” interactivity, and exploring in contextual breadth via self-paced spontaneity. Furthermore, CARE incorporates express tokens for information interchange (eTokin) into the CMD triad to prepare integral content management and informative delivery. In particular, by exploiting eTokin, CARE promotes seamless intercommunications in-between and empowers users to be UNIX professionals cohesively, such as ubiquitous manager on content management and delivery, novel designer on universal interface, intelligent expert for business intelligence, and extraordinary liaison with XaaS without explicitly coding. More CARE uses algorithmic machine learning to coordinate instant online publishing, assemble efficient presentations via wiseCIO to end-users, and aggregate diligent intelligence over DATA for business, education and entertainment (iBEE) through robotic process automation

Student Attitudes Contribute to the Effectiveness of a Genomics CURE

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student\u27s epistemic beliefs to achieving positive learning outcomes

Control of Microstructure and Orientation in Solution-deposited BaTiO₃ and SrTiO₃ Thin Films

Columnar and highly oriented (100) BaTiO3 and SrTiO3 thin films were prepared by a chelate-type chemical solution deposition (CSD) process by manipulation of film deposition conditions and seeded growth techniques. Randomly oriented columnar films were prepared on platinumcoated Si substrates by a multilayering process in which nucleation of the perovskite phase was restricted to the substrate or underlying layers by control of layer thickness. The columnar films displayed improvements in dielectric constant and dielectric loss compared to the fine-grain equiaxed films that typically result from CSD methods. Highly oriented BaTiO3 and SrTiO3 thin films were fabricated on LaAlO3 by a seeded growth process that appeared to follow a standard “two-step” growth mechanism that has been previously reported. The film transformation process involved the bulk nucleation of BaTiO3 throughout the film, followed by the consumption of this matrix by an epitaxial overgrowth process originating at the seed layer. Both BaTiO3 and PbTiO3 seed layers were effective in promoting the growth of highly oriented (100) BaTiO3 films. Based on the various processing factors that can influence thin film microstructure, the decomposition pathway involving the formation of BaCO3 and TiO2 appeared to dictate thin film microstructural evolution

Transcriptional Activation of <i>Mina</i> by Sp1/3 Factors

<div><p>Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of <i>Mina</i> gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the <i>Mina</i> 5′ region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080638#pone.0080638-Okamoto1" target="_blank">[1]</a>. In order to explore the mechanisms regulating <i>Mina</i> gene expression, we set out to molecularly characterize the <i>Mina</i> promoter in the region encompassing these SNPs. We used three kinds of assays – reporter, gel shift and chromatin immunoprecipitation – to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of <i>Mina</i> promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of <i>Mina</i> gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.</p></div

EMSA supershift analysis of Sp1/3 association with the four Sp1/3 binding sites comprising the <i>Mina</i> P1 promoter.

<p>(A) Representative EMSA assay revealing the upper and lower bands of complex 1 to contain Sp1 and Sp3, respectively, and the upper band of complex 2 to contain Sp3. Probes p1–p4 were incubated with EL4 nuclear extracts and antibodies to the indicated targets, prior to analysis by SDS-PAGE. Data are representative of 3 independent experiments. B and C) Chromatin immunoprecipitation (ChIP) analysis of Sp1 (B) and Sp3 (C) binding to the <i>Mina</i> P1 promoter region in EL4 cells. EL4 cell chromatin fragments were immunoprecipitated with antibodies against Sp1 or Sp3 (open bars) or control IgG (filled bars) and analyzed by qPCR. <u>Promoter</u> is the <i>Mina</i> P1-proximal promoter region; <u>Intron 2</u> is located in <i>Mina</i> intron 2, ∼6 kb 3′ of the <i>Mina</i> promoter. Data are from 2 (B) or 3 (C) independent experiments.</p

Reporter assay analysis of a 2 kb <i>Mina</i> promoter fragment.

<p>The transcriptional start site (TSS), labeled 0, is from NCBI (NM_025910.3). Internal control plasmid pRL-TK encoding renilla luciferase was cotransfected with each firefly luciferase construct. Parental (light gray filled arrow) and nested deletion constructs are diagrammed to the left of the bar graph with numbers representing the 5′ (and in the case of −64/+80, the 3′) termini of each construct. The inferred positions of P1 and P2 are labeled and depicted as dark gray rectangles. <u>Vector</u> is PGL3 basic vector; <u>SV40 pro</u> is PGL3 basic vector containing the SV40 promoter; and <u>SV40 pro+enh</u> is PGL3 basic vector containing the SV40 promoter and enhancer elements. Data are expressed as relative light units (the ratio of firefly to renilla luciferase activity) and presented as the mean and SEM of three independent experiments.</p

Pharmacologic and siRNA analysis of Sp1/3 activation of <i>Mina</i> promoter activity in EL4 cells.

<p>(A–D) Mapping the key nucleotides required for Sp1/3 binding to each Sp1/3 binding site (Sp1.1–Sp1.4) in the <i>Mina</i> P1 promoter. For each site, gel shift assays were performed with wild type (WT) and mutant (M1–6) probes (comprising an overlapping set of mutations, underlined), as shown to the right of each gel shift assay image. Nucleotides critical for Sp1/3 binding are bolded. Data are representative of 2 independent experiments. (E) All four Sp1/3 binding sites act additively to activate <i>Mina</i> promoter activity. Cloned into a firefly luciferase reporter construct, the four Sp1/3 sites of a WT <i>Mina</i> P1 promoter (spanning position −64 to +151) were mutated individually or in combinations, as indicated (Xs) in the schematic to the left of the bar graph depicting reporter activity. Data are presented as the mean and SEM from 3 independent experiments. Statistical significance was determined by the student’s t-test (ns, not significant; *p<0.05; **p<0.01). <u>Vector</u> is the PGL3 basic vector; <u>SV40 Pro</u> is the PGL3 basic vector containing the SV40 promoter. (F) Effect of Sp1 inhibitor Mithramycin A on <i>Mina</i> transcript level. EL4 cells were treated for 24 hr with 0, 10 nM, 100 nM, 1 uM Mithramycin A or 1 µM DMSO carrier prior to RNA harvest and analysis of <i>Mina</i> mRNA level by quantitative RT-PCR.</p

Sp1 and Sp3 binding to the <i>Mina</i> promoter in resting CD4+ T cells.

<p>(A) The <i>Mina</i> promoter was marked as an epigenetically active region in resting CD4+ T cells. Chromatin fragments from CD4⁺CD62L^HiCD44⁻CD25⁻ T cells of C57BL/6 mice were immunoprecipitated with antibody to either H3K27me3 or H3K4me3, and then subjected to high-throughput sequencing (ChIPseq). The ChIPseq signals from both anti-H3K27me3 (upper panel) and anti-H3K4me3 (lower panel) are shown with correlations to the <i>Mina</i> locus. The depth of the peaks in the histograms corresponds to the number of reads mapped to the location. A schematic of the <i>Mina</i> locus is shown above the histograms. Each box represents an exon. The region that enriched for H3K4me3 but was devoid of H3K27me3 modifications is enlarged and shown above the <i>Mina</i> locus schematic. (B–C) ChIP assay confirmed the binding of Sp1 and Sp3 to the <i>Mina</i> promoter in EL4 cells. Chromatin fragments from CD4⁺CD62L^HiCD44⁻CD25⁻ T cells of BALB/c mice were immunoprecipitated with antibodies against Sp1 (B, open bars), Sp3 (C, open bars), or their corresponding IgG controls (B–C, filled bars). Promoter: <i>Mina</i> promoter region; Intron 2: an Intron 2 region located ∼6 kb downstream of the <i>Mina</i> promoter.</p